Method and construct for producing graft tissue from an extracellular matrix

ABSTRACT

Using animal tissues as starting materials, a method is described for producing extracellular matrix particulates. The invention includes an embodiment wherein the matrix particulates are applied to collagen scaffolds, which can be seeded with living cells or the particulates may alone be seeded with living cells. Further, the invention encompasses bonding the particulates to collagen foams, or collagen threads made into fabrics or to foams combined with threads. The particulates, with or without scaffolding, can be used as tissues for grafting or as model systems for research and testing. The invention also encompasses the spinning of threads on which the matrix particulates are components and the freeze drying of foams to whose surfaces the matrix particulates are attached.

This application is a continuation application of Ser. No. 09/143,986filed on Aug. 31, 1998, now U.S. Pat. No. 6,051,750 allowed, which inturn is a divisional application of Ser. No. 08/471,535 filed on Jun. 6,1995, now U.S. Pat. No. 5,800,537 which is a continuation-in-part of08/302,087 filed on Sep. 6, 1994, now U.S. Pat. No. 5,893,888 which is acontinuation of 07/926,885 filed on Aug. 7, 1992, abandoned. Thecontents of all of the aforementioned application(s) are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

The use of synthetic materials, such as polyester fiber (Dacron™) orpolytetraflurorethylene (PTFE) (Teflon™) as implants designed to replacediseased or damaged body parts, has been extensive. These materials havehowever, enjoyed limited success. This has been due to the poorbiocompatibility of these materials which among other problems,frequently initiate persistent inflammatory reactions. Additionally, thefailure of the body to integrate these materials, because they do notbreak down and do not lend themselves to remodeling by tissue cells thatmay come into contact with them, causes further problems.

Efforts to use animal or human materials have also been unsatisfactorywhen these materials are crosslinked by formaldehyde or glutaraldehyde,for example. The process of generalized aldehydic crosslinking rendersbiomaterials sufficiently unrecognizable to tissue cells so that normalremodeling and integration are not promoted. Similarly, other types ofchemical processing of animal or human biomaterials, such as extractionwith detergents, or hypertonic buffers or hypotonic buffers can alterthem to the degree that they are toxic to tissue cells and ineffectivein promoting angiogenesis and in stimulating repair and remodelingprocesses needed for the conversion of an implant into a functionalsubstitute for the tissue or organ being replaced.

A third approach has been that of reconstituting tissue and organequivalents from structural matrix components, such as collagen, forexample, that have been extracted, purified and combined withspecialized cells and gelled. The process depends upon interactionsbetween the cells and collagen filaments in the gel that the cellscondense and organize. While tissue-like constructs have been fabricatedand been shown to somewhat resemble their natural counterparts, they donot readily develop the matrix complexity characteristic of the actualtissues they are meant to imitate. See, for example, U.S. Pat. Nos.4,485,096 and 4,485,097, both issued to Eugene Bell on Nov. 27, 1984.

SUMMARY OF THE INVENTION

The present invention relates to a method for producing extracellularmatrix particulates constituting a scaffold to which extracellularmatrix particulates are bound and a fabric having tissue extracellularmatrix particulates.

A tissue source having living cells is processed to derive anextracellular matrix, whereby the living cells are disrupted to formcell remnants. The tissue source is processed to remove the cellremnants from the extracellular matrix of the tissue source withoutremoving growth factors necessary for cell growth, morphogenesis anddifferentiation to form a processed extracellular matrix. The processedextracellular matrix source is fragmented to produce tissue matrixparticulates. Further, the extracellular matrix particulates can becombined with a biopolymer scaffold. The scaffold with extracellularmatrix particulates can be seeded with cultivated cells under suchconditions that the cells adhere to the scaffold and extracellularmatrix particulates.

A biopolymer scaffold consists of a fabric, which is formed of a polymerby weaving, knitting, braiding or felting threads and to which theextracellular matrix particulates are applied. If the polymer iscollagen, the scaffold is crosslinked before the particulates areapplied to it.

An advantage of this invention is that the formed tissues include use asskin, blood vessels, glands, periodontal prostheses and others.

DETAILED DESCRIPTION OF THE INVENTION

The above features and other details of this invention will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Theprincipal features of this invention can be employed in variousembodiments without departing from the scope of the invention. All partsand percentages are by weight unless otherwise specified.

The present invention embodies a new method for preparing animal tissuesand material derived from them as transplants designed to substitute fortissues or organs that are damaged or diseased. This is accomplished insuch a way that their native complexity is preserved and thus allowsthem to be remodeled and integrated by the specialized cells with whichthey are brought into contact. In other words, because the matrixparticulates of the present invention contain cell growth anddifferentiation stimulatory molecules, they provide the biomolecularsignals needed for in vivo tissue repair or for tissue formation invitro in a model tissue system in which cells have been seeded. Incontrast to methods previously described, the method of the presentinvention avoids the use of harsh reagents, such as high salt, ordilapidation reagents, such as butanol/ether or detergents. The use ofsuch reagents in methods previously described is responsible forremoving from the source tissue a great many factors whose presence isconsidered essential for stimulating repair and remodeling processesneeded for the conversion of an implant into a functional substitute forthe tissue or organ being replaced.

The invention includes a method of producing tissues for grafting or foruse as model systems in vitro by obtaining extracellular matrix fromfetal and other tissue sources; by processing it to remove associatedcells without removing factors necessary for cell growth, morphogenesisand differentiation and by combining it with collagen scaffolding withor without cells to produce tissues.

A scaffold or scaffolding includes polymers, such as collagen in theform of a foam, thread, fabric or film. Also, the scaffold can be otherbiodegradable polymers. Further, the scaffold can be the microparticulates themselves.

To prepare the extracellular matrix, the fetuses or other animalmaterials can be flash-frozen at the slaughterhouse. Fetuses are frozenin utero with the ends of the uteri tied off before freezing to insurethe sterility of fetuses in their amnions. The fetuses are thawed underclass 100 clean room conditions and the tissues are dissected forprocessing.

In one embodiment, the tissue is minced, manually and mechanicallywithout heating to rupture the structure and reduce it to particulatesof sizes no greater than about two cubic millimeters. Particles arewashed in buffer to remove blood and intracellular components. Particlesare then freeze-dried, cryomilled and size-sorted. The range of sizes isin the range of between about 10 and 500 μm in diameter. In anotherembodiment, the tissue is freeze-dried after dissection and processed ondemand.

The invention also includes methods of producing extracellular matrixfor tissue building which comprise the above method with the additionalsteps, alone or in combination, of 1) again processing the cellulartissue particulates to remove the components without removing thefactors necessary for cell growth, morphogenesis and differentiation; 2)combining the particulates with collagen scaffolding, and 3) seeding thescaffold to which extracellular matrix particulates are attached withcultivated cells under such conditions that the cells adhere to orpopulate the scaffold.

Advanced methods of in vitro cell cultivation permit expansion of smallnumbers of stem cells or of differentiated cells into large cell banks.Tissue matrix vehicles that can stimulate cell division anddifferentiation of these specialized cells for delivery to the humanrecipient in the clinic are needed. The present invention can be used tobring a disease, such as diabetes, under control by engineering andimplanting reconstituted tissues or composites populated by cells withthe appropriate functional capacity to overcome the problems caused byabsent or insufficient insulin biosynthesis or its regulated delivery.For example, particulates of the present invention can be combined invitro with cells from the islets of Langerhans, or with undissociatedislets, first to stimulate division of the glandular islet cells andsecond to provide transplantation vehicles in the form of pseudoislets.In another embodiment, the pseudoislets can be combined into atissue-like structure by seeding them into collagen foams to create acoherent tissue. An example of a method for forming biopolymer foamshaving extracellular matrix particulates is disclosed in U.S. patentapplication Ser. No. 08/343,172, filed on Nov. 22, 1994, which issued asU.S. Pat. No. 5,709,934 on Jan. 20, 1998, teachings of which are hereinincorporated by reference in its entirety. Further, pseudoislets may beimplanted directly, for example, by using a hypodermic syringe orsimilar devise to deliver them to the desired site. This inventionsimilarly may be used to treat Parkinson's disease by providing thepatient with a tissue containing dopamine producing cells. For that use,nerve cells can be plated onto the particulates to allow a deliveryvehicle to form. This can be useful in the repair of neural tissuedefects.

The method of this invention also has broad applications in therebuilding of damaged or aberrant body structures, such as skin, varioustubular structures and skeletal structures, such as bone, tendon andligament. The invention may also be used to create tissue scaffoldscombined with extracellular matrix particulates without added cellswhich scaffolds, after implantation at a chosen site, are populated bycontiguous or circulating cells of the host. In one embodiment, theextracellular matrix particulates can be added uniformly to the surfacesof a collagen foam cast in a required shape, a sheet or tube, forexample. In another embodiment, the particulates of the presentinvention can be added to collagen fibers or to fabrics of collagenfibers prepared by textile methods in the shape of prostheses. Forexample, the particulates can be used to coat collagen threads or otherbioabsorbable or biodegradable or non-biodegradable polymer threads. Forexample, collagen is extruded into a coagulation bath, then transferredto a bath containing absolute ethanol or acetone, or another dehydratingsolution. The thread can also be subjected to dehydration by some othermeans, for example vacuum-drying. Before coating with extracellularmatrix particulates, it is required that the collagen be crosslinked.The preferred method for crosslinking is by exposure to ultravioletradiation as those practiced in the art are aware. An example of anapparatus and method for spinning and processing collagen fiber havingextracellular matrix particulates is disclosed in U.S. patentapplication Ser. No. 08/333,414, filed on Nov. 2, 1994, which issued asU.S. Pat. No. 5,562,946 on Oct. 8, 1996, the teachings of which areherein incorporated by reference in its entirety.

In a further embodiment, the method of the present invention is used toproduce matrix particulates from fetal animal skeletal elements prior tomineralization or from demineralized animal bone. The fetal matrixparticulates can be combined with osteogenic cells in bone-shapedcollagenous containers to form skeletal constructs. The collagenous,external surface of the containers can be plated with a second cell typeassociated with periosteal tissue. In a similar manner, a compositederived from dermal matrix particulates combined with a foam, or fiber,or foam-plus-fiber sheet can be over-plated with keratinocytes to form askin-like construct.

The cell-free tissue particulates combined with a collagen scaffold madefrom a collagen foam, collagen threads in fabric form or from acombination of foams and threads can be used as an implant for hostcells to populate. Some freeze-dried tissues can also be used withoutfragmentation, but perforated before use.

The invention includes another method for producing tissues whichcomprises obtaining a desired tissue source having an extracellularmatrix and processing as above to remove living cells without removingthe growth factors necessary for cell growth and differentiation. Thetissue source can be frozen or freeze-dried, then fragmented to producetissue particulates. The tissue particulates are seeded with cultivatedcells under such conditions that the cells adhere to and populate thetissue particulates. In one embodiment, the tissue particulates areseeded with cultivated cells and then packed into a preformed porouspolymeric container. This method can also include the optional step offurther processing the tissue particulates before they are seeded, inorder to remove remaining cellular components without removing otherfactors necessary for cell growth and differentiation.

The invention includes another method for producing tissues whichcomprises obtaining a desired tissue source and processing as above toremove living cells without removing factors necessary for cell growthand differentiation. The tissue source is then processed by a method,such as freezing or freeze-drying, and fragmented to produce specifictissue particulates. In the alternative, the cells can be removed priorto fragmentation. The particulates can be applied to threads of collagenor other polymers to make coated threads, or they can be applied tofabrics made from the threads or foams. If the threads or foams orfabrics formed from the threads are collagen, they are crosslinkedbefore adding microparticulates. The threads of the invention are coatedwith particulates which come from a specific tissue or from acombination of tissues. Crosslinking with ultraviolet radiation mustprecede the application of particulates whose biological activity wouldotherwise be adversely affected. These threads are then braided or wovento form fabrics or other complex constructs for implantation or for usein model systems for research and testing applications.

The invention encompasses another method of producing tissue whichcomprises obtaining a desired tissue source and processing to removeliving cells without removing factors necessary for cell growth anddifferentiation. According to this method, the tissue source can beprocessed by freezing or freeze-drying, but not fragmented. Instead, thetissue is processed in the form of sheets or strips. These sheets orstrips can be frozen or freeze-dried, then thawed and perforated toallow cells to infiltrate the tissue. In one embodiment, the tissuesheets or strips are seeded with cultivated cells under such conditionsthat the cells adhere to and populate the sheets or strips. The seededsheets or strips can be further shaped or molded to form useful grafttissue for grafting or for model systems. In another embodiment, thesheets or strips can be implanted into a host without first seeding withcultivated cells. According to this embodiment, the sheets or stripswould provide a site for host cells to populate.

Fragmentation of the desired tissue can be achieved by freezing orfreeze-drying, and then mechanically blending the frozen or freeze-driedtissue or mechanically crushing it between rollers. The desired tissuecan also be fragmented by blending the tissue without first freezing it.An optional nuclease treatment is carried out for the purpose ofdigesting the nucleic acids released when the cells and cell nuclei areruptured, which may result from the treatment of cell removal. Thetissue particulates are then washed and collected by the serial steps ofcentrifugation, resuspension, recentrifugation and resuspension.

The invention further encompasses a method wherein the tissueparticulates obtained by the above methods are layered onto a syntheticporous membrane to a thickness which allows diffusion of nutrients toall the tissue particulates. One embodiment of this method includes anadditional step of bonding or fusing the tissue particulates to form acomposite using a fusing agent. The type of fusing agents which can beselected are known to one skilled in the art. A collagen solution mixedwith particulates and then dried or freeze-dried is an example of afusing agent and process. Various biological glues, as known in the art,are fusing agents. In a preferred embodiment, in vitro cultivated cellsare plated onto the tissue composite in a volume which just cover thecomposite and allow transportation of nutrients. The cells are platedunder conditions that allow the cells to populate or adhere to thetissue particulates. One or more cell type can be plated onto thecomposite.

The invention further includes a construct which is produced by any ofthe above methods, including a multi-cell construct.

The tissue source can be derived from bovine, ovine, porcine or marineanimal tissues, as well as from other species of vertebrates orinvertebrates. The invention further includes extracellular matriceswhich are derived from a variety of body parts or tissues. In apreferred embodiment, extracellular matrix is derived from embryonic orfetal tissues. Embryonic and fetal tissues are advantageous because theyinclude various biomolecular factors which are present in normal tissueat different stages of cell development. They are also initially sterileif prepared by the method described. The factors present in embryonicand fetal tissues include, for example, factors necessary for celldivision, tissue morphogenesis and differentiation.

The term, “factors necessary for cell growth, morphogenesis anddifferentiation”, includes the biomolecular factors which are present innormal tissue at different stages of embryonic and fetal development andduring tissue maintenance and repair throughout life during which celldivision and differentiation may occur continuously or periodically.These factors promote angiogenesis and stimulate repair and processesneeded for the conversion of an implant into a functional substitute fora tissue being replaced or for a tissue being built by cells in vitrofor research and testing applications.

The extracellular matrices of tissues in the body are rich in structuralproteins and in growth and differentiation factors secreted by cells.The structural elements, which include collagens, glycoproteins andproteoglycans, are arrayed in a tissue specific microarchitecture. Thegrowth and differentiation factors, which cells secrete, bind to thestructural elements of the extracellular matrix and regulate cellbehavior in a number of ways. For example, the extracellular matrixcontrols the local concentration of and the biological activity of cellsecreted factors which can then stimulate the secreting cells, exertingan autocrine influence. Alternatively, the secreted factors canstimulate neighboring cells, exerting a paracrine influence. Theinfluence, in the form of gene activation, leads to new biosyntheticproducts or to cell division or cell detachment from a substrate or tocell movement.

The components of the extracellular matrix itself can stimulatebiosynthesis of growth factors and growth factor receptors found at thesurface of cells protruding into the extracellular matrix. Theextracellular matrix can also be mitogenic. The structural proteins ofthe matrix embody intrinsic growth factor activity. For example agrin,laminin, and thrombospondin contain epidermal growth factor (EGF)repeats. Laminin, tenacin and thrombospondin have been shown to exhibitmitogenic activity. The extracellular matrix can control cell divisiontriggered by growth factors by regulating cell shape. Some growth anddifferentiation factors bind to the extracellular matrix by way ofglycosaminoglycans.

Examples of factors which bind to heparin or heparin sulfate chainsinclude epidermal growth factor, multipotential growth factor, mastcell-stimulating factor, platelet-derived growth factor, transforminggrowth factor-β binds to proteoglycan core proteins and glycoproteins,such as fibronectin and thrombospondin, platelet-derived growth factorbinds to osteonectin, and β-endorphin to vitronectin. Using ELISA andradio immune assay, the presence of TGFβ1, 2 & 3, PDGF, FGF and IGF invarious extracellular matrix tissue particulates after cryopreservationand thawing have been demonstrated. This does not suggest that othersare not present; simply, assays for others have not yet been carriedout. Other extracellular components, such as a proteoglycans (e.g.,decorin) and collagen types I and III, have also been demonstrated.

Presently, there is no exact number of “growth factors necessary forcell growth or differentiation or tissue building” because they arecontinually being identified. However, one having ordinary skill in thisart understands and appreciates that these growth factors in cells areattached to structural elements of the extracellular matrix includingproteins, glycoproteins, proteoglycans and glycosaminoglycans. It iswell understood now that the principal activities of cells are governedby signals from the extracellular matrix. The growth factors aresecreted, bind to the extracellular matrix and regulate cell behavior ina number of ways. These factors include, but are not limited to,epidermal growth factor, fibroblast growth factor (basic and acidic),insulin growth factor, nerve growth-factor, mast cell-stimulatingfactor, platelet-derived growth factor, transforming growth factor-β,platelet-derived growth factor, scatter factor/hepatocyte growth factor,Schwann cell growth factor and pleiotrophin depending on the tissuesource.

The tissue source is processed to remove cell contents without removingthe growth factors and structural molecules with which they areassociated in the extracellular matrix and which are necessary fortissue building by cells. The products, extracellular matrixparticulates, are then used in the ways described. In a preferredembodiment, they are combined with a collagen scaffold in the form of acollagen foam or a collagen fabric of threads to constitute a prosthesisof some required shape to which cells may or may not be added. Theminimally processed extracellular matrix is a tissue-specificinformational component to be added to the scaffold of the prostheticdevice. The invention has the advantage of employing tissue componentsfrom a non-recipient and of not being limited to autoimplantation. Forin vitro use as model systems for research for testing or for otherapplications, cells are added to the particulate-decorated scaffolds toconstitute living tissues.

An additional embodiment of using the extracellular matrixmicroparticulates consists of inactivating them by heating to about 56°C. for one hour. While this treatment destroys chemical biologicalactivity, it does not degrade microarchitectural relationships ofcomponents. Active extracellular matrix components are then added to theheat-treated particulates to provide specific single or multipleinstructive signals.

The connective tissue matrix and particulates derived through the abovemethods and combined with a shaped collagen scaffold can be seeded withselected cultured human or other cells. The reconstituted living tissueof various shapes can be used with or without the addition ofsupplementary growth and differentiation factors.

In a preferred embodiment, the matrix particulates are selected to matchthe tissue being fabricated. For example, if a skin replacement is theobjective, preferably dermal matrix would be particularized and with ascaffold, a composite with dermal cells constructed. If neural tissuereplacement is the goal, preferably embryonic or fetal central nervoussystem connective tissue matrix would be chosen for combination with theappropriate cells to form a composite.

The processing steps of the above methods are performed in order toremove cells from the selected animal tissues, thereby producingextracellular matrix reduced to particulates. There are many methodsknown in the art for removing cells from tissue. The following areexamples of some of these methods. These examples are not intended to bean exhaustive list, but are merely examples of some of the knownmethods. The choice of method and the sequence of treatments depend onthe type of tissue being processed.

Various methods for removing cells from the tissue source withoutremoving factors necessary for cell growth, morphogenesis anddifferentiation include scraping tissue with a blade or blade-likeinstrument. Alternatively, the tissue can be squeezed by rollers toremove the soft components including cells. The tissue is not fragmentedwhile removing the cells. The cells are destroyed but not all of thecellular components are necessarily removed even though the cells areruptured. Although a blade, rollers or enzymes can macerate the tissue,the fragmentation step can be separate. For instance, the tissue issqueezed and washed to remove cell fragments. These methods can also beemployed to remove remaining cytoplasmic and nuclear components intissue particulates produced by fragmentation of the tissue source.

The freezing and/or freeze-drying, and fragmentation steps are done toproduce particulates and to further free the tissue of undesirablecomponents. Since cells are fractured by freezing and contents arereleased, the tissue can also be treated with enzymes, particularlynucleases. Additionally, the tissue can be passed through a freeze-thaw,or freeze-dry-thaw cycle in order to disrupt the living cells to formcell remnants which can be washed out of tissues leaving essentiallyextracellular matrix. Methods for sterilizing processed tissues areknown to one skilled in the art and include contacting the tissue with aperacetic acid solution, preferably about a 0.5% solution, and washingthe tissue in a sterile buffer, preferably sterile phosphate bufferedsaline. The tissue can then be freeze-dried by being brought to thetemperature of liquid nitrogen, then fragmented under sterileconditions.

Fetal tissue taken from sterile fetuses contained in their intactamnions can be kept sterile under clean room conditions. Fragmentationat low temperature can be carried out in many ways known to thoseskilled in the art; manual shearing with knives or scissors is one.Fragmentation methods could include a shearing device, such as amechanical blender, or a mechanical crusher to yield particulates.Particulates are further reduced by cryomilling, after which they arescreen-sized. In a preferred embodiment, the particulates fall in arange between about 10 and 500 micrometers in diameter; for someapplications the particulates can be as small as about 5-10 microns.Depending on the starting tissue type, further nuclease cleaning andwashes followed by centrifugation and resuspension in fresh phosphatebuffered saline can be carried out in an effort to minimize the presenceof components which could be immunogenic.

In another embodiment, the tissues can be dehydrated chemically withacetone, absolute alcohol or hydrophilic polymers, such as carbowax, andthen fragmented.

After the particulates are prepared, they are seeded with cells.Preferably, one can use human cells for populating the matrix, but theinvention is not limited to human cells. In one embodiment, the seedingprocess is carried out by rehydrating the matrix particulates usingtissue culture medium, with or without growth and differentiationsupplements added, and allowing the particulates to swell to equilibriumwith their fluid environment before applying cells to them.

In another embodiment, the matrix particulates are populated with cellsof one, or more than one phenotype in a bioreactor, spinner flask orother similar device to expand cell number. The number of cells used andthe time of residence in the device depends on cell attachment time andthe desired degree of coverage of the particulate by the cells. Theseparameters vary depending on the type of cell used and the cell densityrequired for the final product. The particular parameters for each celltype will be apparent to one skilled in the art. In a preferredembodiment, the starting cell density is in the range of between about0.5×10⁴ and 10⁶ cells per milliliter. In another embodiment, theprepared particulates can be combined with collagen scaffolds in theform of foams or fiber based textiles of shapes appropriate forprostheses or model systems for research or testing applications.

The matrix particulates, with or without cells, can be layered onto asynthetic porous membrane. Examples of a synthetic porous membrane, forillustrative purpose only and not to limit the invention in any way,include membranes made of polycarbonate, for example, in a transwellarrangement, or onto a bioabsorbable polymer, such as a porous collagenfoam that lines a mold having a porous membrane for a bottom to hold thecollagen foam and permit passage of medium from below. The membrane mayalso be made of other bioabsorbable polymers, such as poly-1-lactate orof thermosetting polymers. In one embodiment, the particulates arelayered in a single layer. Also, in one embodiment, the layer may varybetween about 0.2 and 3 millimeters in thickness.

A further embodiment includes the use of multiple cell types, one typeplated in a collagen foam to which matrix particulates are applied or oncollagen threads which have been decorated with adhering matrixmicroparticulates and another cell type plated on the lower or uppersurface of the scaffold.

Further, the invention encompasses the embodiment where the cell-ladenparticulates are packaged in a preformed porous collagen or otherpolymeric container of any desired shape. Additionally, the inventionincludes the use of concentric tubes of differing radii where, in apreferred embodiment, the radii would differ by only about onemillimeter or less. It is also envisioned that the other shapes can beused as well.

The containers can be reinforced by struts or other means to maintainuniform thickness. The frequency of the struts is preferably in therange of between about one and ten per square millimeter. The containercan be made of a collagen sheet with pores that are too small to allowparticulates to escape. In a preferred embodiment, the pores measure inthe range of between about ten and sixty microns. After introduction ofparticulates through an end opening, the container can be sealed bysuturing or other means.

In another embodiment, the tissue particulates are applied to threadssuitable for weaving or braiding. If the threads are of collagen, theyare crosslinked before addition of matrix particulates which adhere tothe threads when applied after the crosslinked threads are hydrated. Thethreads can be woven into fabrics or other complex constructs forimplantation. The matrix particulates can be applied to the fabric afterthey are braided, woven, etc. The braided or woven fabrics provide ascaffold with applied matrix particulates for host cells to adhere to.In addition, the thread or fabrics produced from the threads can beseeded with cells in vitro, as above, and then transplanted into theappropriate site in the host.

In another form, a tissue can be constituted from a collagen foam towhose surfaces the particulates are applied. After a foam is formed byfreeze-drying a volume of collagen in solution, the foam is crosslinkedusing ultraviolet radiation, a procedure known to those practiced in theart.

The foam is then filled with a collagen solution containing theextracellular matrix particulates and freeze-dried again, but notcrosslinked. The result is that the surfaces of the foam are coated withthe matrix particulates. The polymer used for producing the threads canbe either bioabsorbable or non-bioabsorbable. In a preferred embodiment,the polymer is collagen. After extrusion, the threads of this inventionare coagulated and dehydrated, for example, in a bath containingabsolute alcohol, carbowax or acetone. The threads can then be dried andspooled, for example, by pulling the moving thread over more rollers,stretching and drying it and then winding it onto spools.

EXEMPLIFICATION I

For example, to reconstitute a skin substitute, a sheet of collagen foamenclosing a collagen thread fabric reinforcement at one of its surfacesis crosslinked with ultraviolet light and filled with a solution ofcollagen at 1.0 μg/ml containing 2 mg/ml microparticulates derived fromnear-term fetal dermis. The foregoing combination is freeze-dried over a60-hour period so that all surfaces of the collagen scaffolds are coatedwith microparticulates.

The resulting biologically active scaffold is seeded with cultivateddermal fibroblasts at 10⁵ cells/ml introduced into the foam side of thesheet. The threaded fabric side is plated with cultivated keratinocytesat 10⁶ cells/ml. The cell-seeded scaffolds are incubated in a 10% CO₂incubator for 21 days to produce a differentiated skin prosthesis.

EXEMPLIFICATION II

An alternative includes of using a double-density foam sheet bonded tothe single density foam at one surface of the single density foaminstead of incorporating the collagen thread fabric as in Example 1. Thedouble-density foam is produced by hydrating a crosslinkedsingle-density foam and allowing it to dry at a temperature not greaterthan about 37° C. The sheet of double-density foam is positioned on thelower surface of a rectangular mold the size of the finished skinproduct. The double-density mold is hydrated and the mold is filled witha collagen solution of 5 mg/ml and freeze-dried. The single-density foamnow fused to the double-density foam is crosslinked and dermis-specificextracellular particulates are added to the scaffold as described inExample 1. Cultivated dermal fibroblasts are seeded into thesingle-density foam and cultivated keratinocytes are seeded onto thedouble-density foam. The skin prosthesis is incubated as described inExample 1.

EXEMPLIFICATION III

To reconstitute an endocrine pancreas equivalent at least three organscan be used. The first is the pancreas itself, which would provide thetissue specificity of the matrix. The second is the duodenum or jejunumof the gut, and the third is the skin.

The organs are packed on ice in sterile containers immediately afteranimal slaughter and transported to the laboratory. The pancreas istreated using the protocol for islet removal, that is, the pancreaticduct is cannulated and Hank's solution containing 2 mg/ml collagenase(Type X, Sigma) and 2% fetal calf serum is injected. In addition, thetwo arteries that supply the pancreas, the celiac and the superiormesenteric, are similarly perfused with a collagenase solutioncontaining heparin to remove endothelium from the circulatory tree ofthe gland. Adequate cleaning of the pancreas may require heparinizinganimals before slaughter. A compression device is used to gently kneadthe organ as it is perfused and reperfused in a sterile chamber in thecold.

In the course of recirculation, the collagenase solution is graduallydiluted. A fibrous network of ducts and vessels remains aftercollagenase digestion. After washing in phosphate buffered saline, thefibrous material is freeze-dried, sheared in the cold and cryomilled.

The objective of the freezing and fracturing procedure is to providematrix particulates of between about 50 and 100 μm in diameter. The sizeof the particulates is also determined by the maximum allowablethickness of a tissue graft made to a host. The thickness must beconsistent with survival after implantation in vivo. A collagen foam isa preferred vehicle for transplanting islets or pseudoislets because ofits porosity, which include microcompartments of sizes suitable forhousing islets and because it is rapidly vascularized. The goal is toproduce particulates of relatively uniform size from the frozen tissueby a mechanical process that does not melt the material by frictionalheating. Blending, grinding, crushing, and/or percussion in the cold at−30° C. or below are methods which can be used.

The particulates are then combined into aggregates with small clustersof cultivated cells, or are used as microbeads in a cell bioreactor inwhich cells from islet cell cultures are seeded. The cells attach tosurfaces of the matrix microbeads in the bioreactor. The last mentionedstrategy is useful as a means of expanding islet cell cultures; it canalso serve as a means for constituting pseudo-islets.

The second example of an organ processed for its matrix is the jejunumor duodenum which is mechanically scraped after opening. As described inU.S. Pat. No. 4,902,508, issued to Badylak et al. on Feb. 20, 1990, thestratum compactum, about 100 μm thick, lying between luminal layers onone side and the muscular layers on the other, is delaminated from them,leaving an acellular collagenous connective tissue. The washed stratumcompactum is particularized after freeze-drying, as described above.

Similarly, the third organ, skin, is stripped of epidermis andunderlying adipose material and the dermis is sectioned into 100-200 μmthick sheets parallel to the dermal surface with a dermatome. It is thenfreeze-dried, particularized and washed in preparation for combinationwith cells or transplantation. Alternatively, the sheets derived fromthe jejunum and the dermis can be made porous or perforated with ameshing machine or similar device so that they can be seeded with cellsor pseudo-islets or both.

Tissue can be formed by combining cells with foams, threads, or foamsand threads to which generic or tissue-specific extracellularparticulates have been applied. Such tissues can be in the form ofprostheses for grafting or model systems for use in basic and appliedresearch and for testing and diagnostic purposes. The tissues can bemade to fit into transwell chambers, such as those produced by suppliersof tissue culture ware.

Equivalents

Those skilled in the art will know, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. These and all otherequivalents are intended to be encompassed by the following claims.

What is claimed is:
 1. A method for producing extracellular matrixparticulates, which comprises the steps of: a) having an extracellularmatrix and living cells; b) processing the animal tissue to disrupt theliving cells to form cell remnants; c) separating and removing the cellremnants from the extracellular matrix without removing growth factorsresponsible for regulation of cell growth, morphogenesis anddifferentiation to form a processed extracellular matrix; d) fragmentingthe processed extracellular matrix to produce extracellular matrixparticulates; and e) optionally processing the extracellular matrixparticulates further to remove additional cell remnants without removinggrowth factors responsible for regulation of cell growth, morphogenesisand differentiation, wherein the extracellular matrix particulates arecombined with a collagen scaffold for use as a tissue substitute.
 2. Themethod of claim 1 wherein the scaffold is selected from the groupconsisting of a thread, fabric, and foam.
 3. The method of claim 1wherein the extracellular matrix particulates with scaffold are seededwith cultivated cells under such conditions that the cells adhere to theextracellular matrix particulates and scaffold.
 4. The method of claim 1wherein the animal tissue includes embryonic or fetal tissue.
 5. Themethod of claim 1 wherein the fragmenting step comprises mechanicallycutting up the animal tissue that is frozen or unfrozen.
 6. The methodof claim 1 wherein the fragmenting step comprises mechanical crushingbetween rollers.
 7. The method of claim 1 wherein the fragmenting stepis followed by enzymatic treatment to remove nucleic acids.
 8. Themethod of claim 1 wherein the extracellular matrix particulates arefused by a fusing agent to form a composite.
 9. The method of claim 3wherein the cultivated cells are suspended and plated onto a compositein a volume which cover the composite and allow transportation ofnutrients, under such conditions that the cells populate theextracellular matrix particulates.
 10. The method of claim 9 wherein thecells include more than one cell type.
 11. The method of claim 1,further comprising the steps of freezing or freeze-drying theextracellular matrix before fragmenting the extracellular matrix,wherein the extracellular particulates are applied to a surface of thescaffold upon combining the extracellular matrix particulates with thescaffold, and wherein the scaffold is cross-linked.
 12. An extracellularmatrix particulate produced by the method of claim
 1. 13. A constructproduced by the method of claim
 2. 14. A construct produced by themethod of claim
 3. 15. The method of claim 11, wherein the cell remnantsare cytoplasmic and nuclear components.
 16. The method of claim 11,wherein the tissue substitute is a graft.
 17. The method of claim 4wherein the embryonic or fetal tissue is selected from the groupconsisting of bovine, porcine and marine animals.
 18. A tissuesubstitute produced from the method of claim
 1. 19. A method forproducing an unfragmented, processed extracellular matrix constructwhich comprises the steps of: a) isolating animal tissue having anextracellular matrix and living cells; b) processing the animal tissueto disrupt the living cells to form cell remnants without fragmentingthe tissue; and c) separating and removing the cell remnants from theextracellular matrix without removing growth factors responsible forregulation of cell growth, morphogenesis and differentiation and withoutfragmenting the tissue to form the unfragmented, processed extracellularmatrix construct for use as a tissue substitute.
 20. The method of claim19 wherein the processing, separating and removing steps comprisefreezing or freeze-drying the tissue.
 21. The method of claim 19 whereinthe extracellular matrix construct is seeded with cultivated cells undersuch conditions that the cells adhere to the extracellular matrixconstruct.
 22. A method of using the tissue subsitute as in claim 1 bygrafting the tissue substitute to a recipient.